Process for detecting the expression of CD95 ligand in cells

ABSTRACT

The present invention relates to a process for quantitatively determining CD95 ligand, comprising the following processing steps: 
     (a) isolation of total RNA from cells, 
     (b) transcription the total RNA from (a) into cDNA by reverse transcription, and 
     (c) amplification of the cDNA from (b) and a CD95 ligand competitor fragment by CD95 ligand-specific primers in a PCR reaction. 
     The process is suitable for determining the extent and/or course of apoptosis.

This is a national phase filing of the Application No. PCT/DE96/02274,which was filed with the Patent Corporation Treaty on Nov. 27, 1996, andis entitled to priority of the German Patent Application P 195 44 332.2,filed Nov. 28, 1995.

FIELD OF THE INVENTION

The present invention relates to a process for detecting the expressionof CD95 ligand in cells.

BACKGROUND OF THE INVENTION

Apoptosis is the designation for programmed cell death. It is found e.g.in organogenesis and metamorphosis, tissue atrophy and tumor regression.Apoptosis is linked with a condensation of the cytoplasm, a loss ofplasma membrane villi, a segmentation of the nucleus and extensivedegradation of chromosomal DNA. It has turned out that disturbances ofapoptosis occur in various diseases such as AIDS, autoimmune diseases,tumors and leukemias. In the case of AIDS, increased apoptosis seems tobe responsible for the strong decrease of the CD4-T cells.

In activated T cells and in other cells there is found a cell surfaceprotein referred to as CD95 and APO-1, respectively. A soluble ormembrane-bound protein referred to as CD95 ligand and APO-1 ligand,respectively, can bind to this cell surface protein and trigger theinduction of apoptosis.

The extent and course of apoptosis as well as the molecules causing thesame cannot be determined so far. However, this would be necessary to beable to carry out e.g. follow-ups and suitable therapeutic measures inpatients suffering from the above-mentioned diseases.

Therefore, it is the object of the present invention to provide aproduct serving for determining the extent of apoptosis and its course,respectively.

SUMMARY OF THE INVENTION

The present invention relates to a process for quantitativelydetermining CD95 ligand, comprising the following processing steps:

(a) isolation of total RNA from cells,

(b) transcription the total RNA from (a) into cDNA by reversetranscription, and

(c) amplification of the cDNA from (b) and a CD95 ligand competitorfragment by CD95 ligand-specific primers in a PCR reaction.

The process is suitable for determining the extent and/or course ofapoptosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the quantitative PCR according to theinvention for CD95-L.

FIG. 2 shows a diagram for the preparation of a competitor DNA fragmentof CD95-L.

(A) The wild-type fragment and its specific primers for PCRamplification, L_(up) and L_(down), are shown.

(B) L_(up) and L_(upOV) are used for the 1^(st) PCR. L_(upOV) binds inthe middle of the sequence and contains an overhanging sequence at the5' end having a length of 50 bp.

(C) L_(down) and L_(downOV) are used for the second PCR. The last 30 bpat the 5' end of L_(downOV) are overhanging and complementary to thelast 30 bp at the 5' end of L_(upOV). The complementary regions areshown in brush-shaped fashion.

(D) shows the PCR products from the first and second PCRs, 349 and 230bp long DNA pieces. Following purification, both fragments arehybridized with each other via their complementary regions.

(E) The fragments hybridized in (D) are used as a matrix for a third PCRreaction where L_(up) and L_(down) are used again as primers.

(F) The result is a mutated fragment of CD95-L which carries a 50 bplong insert in the middle of the sequence.

FIG. 3 shows a constitutive and induced expression of CD95-L mRNA inlymphoid cells.

DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide a product servingfor determining the extent of apoptosis and its course. According to theinvention this is achieved by the subject matters defined in the claims.

Therefore, the subject matter of the present invention relates to aprocess by which the CD95 ligand, hereinafter referred to as CD95-L, canbe determined quantitatively in cells.

The present invention is based on the applicant's finding that apoptosisis caused in T cells by an increased amount of CD95-L. Furthermore, theapplicant discovered that the apoptosis rate is determined by the CD95-Lamount.

The process according to the invention relates to a quantitative"polymerase chain reaction" (PCR) by which the amount of CD95-L in cellscan be determined reliably. The amount of CD95-L mRNA present in thecell is determined in the process according to the invention by anRT-PCR reaction. In the quantitative PCR for CD95-L, the amount ofCD95-L cDNA is titrated by adding a CD95-L-specific DNA competitorfragment to the PCR reaction. The complete reverse transcription of adefined amount of total RNA into cDNA precedes this step. The competitorfragment differs from the "wild-type" cDNA of CD95-L by additional basepairs in the middle of the nucleotide sequence but has identicalflanking sequences. Therefore, wild-type and competitor fragment arerecognized by the same primer combination and can be multiplied jointlyin a PCR. Due to the identical primer binding sites and the similarnucleotide sequences, both fragments are subject to the sameamplification rate. However, the minor difference in size permits theseparation of both DNA species by gel electrophoresis according to PCR.The intensities of the DNA bands on agarose gel reflect the originalamounts of cDNA used in PCR. If the results of CD95-L and competitor DNAbands on the gel are identical, it can be concluded that the sameinitial concentrations of competitor and CD95-L were present. The amountof CD95-L cDNA titrated in this way reflects the amount of CD95-L mRNAin the total RNA, since the reverse transcription proceeds fully. Thediagram of the quantitative PCR reaction is shown in FIG. 1.

The quantitative PCR is based on the following reaction principle. Forquantifying the CD95-L expression rate, cDNA of a certain amount ofreversely transcribed total RNA is pipetted into PCR vessels. Competitorfragment in various dilutions, e.g. from 5 to 10000 fg, is added to eachsample with the exception of the negative control (-). Following the PCRreaction, the amplified DNA fragments are treated with ethidium bromideand separated on an agarose gel. The DNA is made visible by irradiatingthe gel with U.V. light. The titration point (in FIG. 1 marked by anarrow) is determined, with respect to which both the bands of the CD95-LDNA and the bands of the competitor DNA have equal intensities. An equalratio of both bands shows that equal amounts of DNA to be determined andcompetitor DNA have been amplified. Correspondingly, the amount ofCD95-L cDNA determined by the titration point (to be equated with theamount of mRNA) had been present in the employed amount of total RNA.

The primers synthesized for PCR are based preferably on the publishedsequence of CD95-L (cf. Takahashi T. et al., Int. Immunol. 6, (1984),1547-1567). PCR is carried out under conditions with which a personskilled in the art is familiar.

For example, the following primers can be used for amplifying the CD95-Lfragment (about 500 bp) and the competitor fragment (about 550 bp)

L_(up) 5'-ATGTTTCAGCTCTTCCACCTACAGA-3' (SEQ ID NO: 1)(25 mer, binds tonucleotides 301 to 325 of the CD95-L sequence)

L_(down) 5'-CCAGAGAGAGCTCAGATACGTTGAC-3' (SEQ ID NO: 2) (25 mer, bindsto nucleotides 775 to 799 of the CD95-L sequence)

Furthermore, e.g. the following primers can be used for the preparationof the competitor fragment (550 bp)

L_(upOV) 5'-GGATCCGTACTACAGTGAAATTATGGAAGGGTATCCGAGTTCAGG-AATTCCAGAGGCATGGACCTTGAGTTGGACTTGCC-3' (SEQ ID NO: 3) (80 mer, binds tonucleotides 451 to 480)

L_(downOV)5'-CCTTCCATAATTTCACTGTAGTACGGATCCGAATGGGAAGACACCTATGGAATTGTCC-3' (SEQ IDNO: 4) (58 mer, binds to nucleotides 481 to 508)

The mutated CD95-L fragment which is used in PCR as competitor fragment,is prepared by mutagenesis, preferably PCR mutagenesis. CD95-L CDNAoriginating e.g. from T cell line CEM-S serves as a matrix for PCRmutagenesis. Two parts of the L_(up) /L_(down) PCR fragment areamplified by using in a PCR the primer pair L_(up) /L_(upOV) (yields afragment having a length of 230 bp) and in a second PCR the primer pairL_(down) /L_(downOV) (yields a fragment which has a length of 349 bp).The primer L_(upOV) contains a 50 bp long overhanging sequence which iscomplementary to a 30 bp long overhanging sequence of the primerL_(downOV). Following purification, the two fragments are hybridized viatheir complementary regions and used as a matrix for another PCRamplification with primers L_(up) /L_(down). The result is a mutatedfragment of CD95-L which contains 50 additional bases in the middle ofthe nucleotide sequence. The diagram for the preparation of thecompetitor fragment is illustrated in FIG. 2.

The process according to the invention, which is based on a competitionbetween a mutant fragment and a wild-type fragment, enables an accuratedetermination of the intracellular level of CD95-L in small amounts ofsample (1 to 5×106 cells).

Since the apoptosis conveyed by CD95/CD95-L obviously takes part in Tcell depletion in the case of an AIDS disease, the quantitativedetermination of the expression of CD95-L can be used for the follow-upand as an indicator of therapeutic interventions in AIDS patients.Furthermore, the process according to the invention is suitable in vitrofor "drug screening" for potential drugs which can block virus-induced,CD95-conveyed apoptosis in T cells. Likewise, the quantitativedetermination of the CD95 ligands in cells can be used for the diseasedefinition, follow-up and the pharmacological influence of ligandexpression in "drug screening" in the case of diseases accompanied byreduced apoptosis (e.g. leukemias) or increased CD95-conveyed apoptosis.A direct field of application is represented e.g. by the analysis of theeffect of drugs, such as azothioprin, cyclosporin A, cyclophosphamide,cortisone and methotrexate, which are presently used for theimmunosuppressive treatment of autoimmune diseases and transplantrejections.

The below examples explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

EXAMPLES A. Example 1 Determination of CD95-L in Lymphoid Cells

The lymphoid T cells J16 and CEM-S were cultivated in common broth eachand treated with PMA (5 ng/ml) as well as ionomycin (2 μg/ml),respectively, for 4 hours and 24 hours, respectively. Total RNA wasobtained from the cells according to the method by Chomczynski, P. andSacchi, N., Anal. Biochem., 162, (1987), 156-159 and transcribed bymeans of AMV reverse transcriptase (Amersham, Braunschweig, FRG) andoligo-dT16 primers (Promega, Heidelberg, FRG) into cDNA. A 20 μlreaction comprised 1× AMV buffer (50 mM TrisHCl, pH 8.3; 8 mM MgCl₂ ; 50mM NaCl; 1 mM DTT), 250 μM of each dNTP (Pharmacia, Freiburg, FRG), 0.5μM oligo-dT₁₆ ; 0.6 (U/μl RNase inhibitor (Amersham, Braunschweig, FRG);0.75 U/μl AMV reverse transcriptase; 50 ng/μl total RNA (prior to theaddition denatured at 65° C. for 5 minutes). Reverse transcription wascarried out at 42° C. for 45 minutes, and the reaction was thenterminated by heating to 94° C. for 5 minutes. Since all reagents werepresent in excess, it can be assumed that the RNA employed has beentranscribed completely into cDNA.

For quantifying the CD95-L expression rate, cDNA of 250 ng of reverselytranscribed total RNA each was pipetted into PCR vessels. Competitorfragment, as described above, was added in differing dilutions of 10 to10000 fg to each sample with the exception of the negative control (-).The reaction mixture comprised: 50 μl, containing cDNA (=250 ng ofreversely transcribed total RNA), 1× PCR buffer (10 mM Tris-HCl, pH 8.8;50 mM KCl, 0.08% NP4O), 1 mM MgCl₂, 200 μM dNTPs, 0.5 μM primers and1.12 U/vessel Taq polymerase (MBI/Fermentas). The amplification wascarried out in a robocycler (Stratagene) or a Biozym thermocycler usingthe L_(up) and L_(down) primers as described above. The reactionconditions were as follows: 35 seconds at 94° C., 90 seconds at 56° C.and 120 seconds at 72° C. over 36 cycles. 20 μl of the PCR reaction wereseparated by electrophoresis on a 2% agarose gel and, following ethidiumbromide staining, were made visible by U.V. light. The point at whichboth the bands of the CD95-L DNA and the bands of the competitor DNA hadequal intensities was determined on the gel. The gel is shown in FIG. 3.Equal band intensities which indicate identical amounts of wild-type andcompetitor fragment, are marked by arrows.

It showed that untreated J16 cells expressed 10 fg CD95-L mRNA/250 ngtotal RNA and untreated CEM-S cells expressed 50 fg CD95-L mRNA/250 ngtotal RNA. A PMA/ionomycin treatment increased the expression of CD95-LmRNA to 1000 fg/250 ng of total RNA in the case of J16 cells and to 5000fg/250 ng of total RNA in the case of CEM-S cells (cf. Table 1).

The above data show that CD95-L can be determined quantitatively by theprocess according to the invention. Thus, the extent and/or the courseof apoptosis, particularly in the case of diseases such as AIDS, can bedetermined.

                  TABLE 1                                                         ______________________________________                                        Expression of CD95-L mRNA (fg/250 ng total RNA)                                 in lymphoid cell lines                                                                    CO         4 h P/I 24 h P/I                                     ______________________________________                                        T cell    J 16    10         1000  1000                                         lines CEM-S 50 5000  250                                                    ______________________________________                                    

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 4                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 1                                                         - - atgtttcagc tcttccacct acaga          - #                  - #                   25                                                                     - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 2                                                         - - ccagagagag ctcagatacg ttgac          - #                  - #                   25                                                                      - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 80                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 3                                                         - - ggatccgtac tacagtgaaa ttatggaagg gtatccgagt tcaggaattc ca -             #gaggcatg     60                                                                 - - gaccttgagt tggacttgcc            - #                  - #                      - # 80                                                                  - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 58                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Primer                                                - - <400> SEQUENCE: 4                                                         - - ccttccataa tttcactgta gtacggatcc gaatgggaag acacctatgg aa - #ttgtcc           58                                                                      __________________________________________________________________________

What is claimed:
 1. A process for quantifying a CD95 ligand,comprising:(a) isolating RNA from cells; (b) transcribing said RNA ofstep (a) into cDNA by reverse transcription; (c) amplifying said cDNA ofstep (b) and a defined amount of CD95 ligand competitor fragment usingCD95 ligand-specific primers, wherein the CD95 ligand-specific primershave the following sequences: L_(up) 5'-ATGTTTCAGCTCTTCCACCTACAGA-3'(SEQ ID NO: 1) and L_(down) 5'-CCAGAGAGAGCTCAGATACGTTGAC-3' (SEQ ID NO:2), so that a CD95 ligand amplification product of the cDNA of step (b)and a CD95 ligand competitor amplification product are generated; and(d) quantifying said CD95 ligand by comparing the quantity of CD95ligand amplification product with the quantity of CD95 ligand competitoramplification product.
 2. The process of claim 1, wherein the cellsoriginate from an established cell line or a patient.
 3. The process ofclaim 2, wherein the cells are lymphoid T cells.
 4. A method fordetecting apoptosis, comprising:(a) isolating RNA from T cells; (b)transcribing said RNA of step (a) into cDNA by reverse transcription;(c) amplifying said cDNA of step (b) and a defined amount of CD95 ligandcompetitor fragment using CD95 ligand-specific primers, wherein the CD95ligand specific primers have the following sequences: L_(up)5'-ATGTTTCAGCTCTTCCACCTACAGA-3' (SEQ ID NO: 1) and L_(down)5'-CCAGAGAGAGCTCAGATACGTTGAC-3' (SEQ ID NO: 2), so that a CD95 ligandamplification product of the cDNA of step (b) and a CD95 ligandcompetitor amplification product are generated; (d) quantifying saidCD95 ligand by comparing the quantity of CD95 ligand amplificationproduct with the quantity of CD95 ligand competitor amplificationproduct, wherein an increased quantity of CD95 ligand compared tocontrol T cells is indicative of apoptosis.
 5. The method of claim 4,wherein the T cells in step (a) are from an AIDS patient.